In-Depth Structure and Function Characterization of Heterogeneous Interchain Cysteine-Conjugated Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs), integrating high specificity of antigen-targeting antibodies and high potency of cell-killing chemical drugs, have become one of the most rapidly expanding therapeutic biologics in oncology. Although ADCs were widely studied from multiple aspects, overall structural elucidation with comprehensive understanding of variants is scarcely reported. Here, for the first time, we present a holistic and in-depth characterization of an interchain cysteine-conjugated ADC, focusing on conjugation and charge heterogeneity, and in vitro biological activities. Conjugation mapping utilized a bottom-up approach, unraveled positional isomer composition, provided insights into the conjugation process, and elucidated how conjugation affects the physicochemical and biological properties of an ADC. Charge profiling combined bottom-up and top-down approaches to interrogate the origin of charge heterogeneity, its impact on function, and best practice for characterization. Specifically, we pioneered the utilization of capillary isoelectric focusing-mass spectrometry to decode not only critical post-translational modifications but also drug load and positional isomer distribution. The study design provides general guidance for in-depth characterization of ADCs, and the analytical findings in turn benefit the discovery and development of future ADCs.

Early quantitative infrared pupillometry for prediction of neurological outcome in patients admitted to intensive care after out-of-hospital cardiac arrest.

BACKGROUND: Quantitative pupillometry is recommended for neuroprognostication after out-of-hospital cardiac arrest 72 h or more after ICU admission, but the feasibility and utility of earlier assessment is unknown. METHODS: This was a study of the utility of an early quantitative pupillometry index in predicting neurological outcome in patients with reduced consciousness after out-of-hospital cardiac arrest. Quantitative infrared pupillometry index was measured at 0, 6, 24, 48, and 72 h from admission. Acceptable predictive utility was defined as a positive predictive value of >95% and false positive rate of zero, with a narrow 95% confidence interval (95% CI). RESULTS: At least one quantitative pupillometry index measurement was available from within the first 6 h for all 77 patients who met inclusion criteria. A quantitative pupillometry index ≤2.4 at baseline and ≤2.3 within the first 6 h met the criteria for utility. The positive predictive value of the baseline cut-off (≤2.4) for poor neurological outcome was 1.00 (95% CI, 0.54-1.00) with an estimated false positive rate of 0% (95% CI, 0-9%). The positive predictive value of the 6 h cut-off (≤2.3) for poor neurological outcome was 1.00 (95% CI, 0.59-1.00) with an estimated false positive rate of 0% (95% CI, 0-8%). Sensitivities of these cut-offs for ruling out poor neurological outcomes at 0 and 6 h were 19% and 22%, respectively. Of seven patients with a quantitative pupillometry index ≤2.3 within 6 h of ICU admission, none survived. Analyses that used quantitative pupillometry index measurements from 24 to 72 h, but excluded baseline and 6 h values, were not predictive by the utility criteria. CONCLUSIONS: Quantitative pupillometry within 6 h of ICU admission after out-of-hospital cardiac arrest may identify patients with a very low chance of neurologically intact survival. Further studies of early quantitative pupillometry in this population are warranted.

Development and validation of ion-pairing HPLC-CAD chromatography for measurement of Islatravir's phosphorylated intermediates.

Biocatalytic processes have become more prevalent in the pharmaceutical industry, leading to analytical challenges not faced when characterizing more traditional synthetic routes. A novel one-pot biocatalytic process has been established for Islatravir, an HIV reverse transcriptase translocation inhibitor for the treatment and prevention of HIV-1. As a one-pot reaction, the Islatravir chemistry contains multiple intermediates that are not isolated. Additionally, these unisolated intermediates have no chromophores, making traditional LC-UV techniques ineffective for characterization. A hydrophilic interaction chromatography (HILIC) method with a charged aerosol detector (CAD) was initially developed, however numerous inorganic species present in the one-pot reaction were retained; this led to co-elution of compounds and poor peak shapes. An innovative ion-pairing LC method was developed in order to resolve inorganic species, intermediates, and the API, for use during in-process control of the Islatravir biocatalytic reaction. Aided by a volatile ion-pairing reagent compatible with the CAD, this method successfully retains and resolves the highly polar intermediates of interest and Islatravir API. This novel method was successfully validated and has allowed the Islatravir biocatalytic process to be fully characterized from the early intermediates through the final API within the one-pot reaction without the need for isolations. This novel ion-pairing HPLC-CAD technique lays the groundwork for method development on current and future biocatalytic-produced drug substances.

A preliminary investigation into theory of mind and attributional style in adults with grandiose delusions.

INTRODUCTION: A preliminary cognitive model of grandiose delusions has been put forward suggesting that persecutory and grandiose delusions shared distinct, yet overlapping psychological processes. This study aims to test this model and hypothesises that participants experiencing grandiose delusions may demonstrate a theory of mind (ToM) impairment and differences in attributional style compared to a control group. METHODS: A cross-sectional design compared the performance of 18 individuals with grandiose delusions to a control group of 14 participants with depression. ToM was measured using a non-verbal joke appreciation task and a verbal stories task. Attributional style was measured using the internal, personal and situational attributions questionnaire. RESULTS: Participants experiencing grandiose delusions performed significantly worse on both ToM tasks compared to controls. Furthermore, these participants provided significantly more atypical answers when explaining the joke behind the ToM cartoons. No differences for subjective funniness ratings or attributional style were found. CONCLUSIONS: This preliminary study indicated participants experiencing grandiose delusions have ToM impairments which may contribute to the maintenance of this symptom.

A simple parallel gas chromatography column screening system.

A simple approach to the automated screening of four different columns on a single gas chromatography (GC) instrument is used for rapid chiral GC method development. Configuration of a conventional GC instrument with a second autosampler and several inexpensive Y-splitters enables simultaneous evaluation of two different columns, allowing a total of four different columns to be evaluated in two automated back to back runs. The resulting system affords a simple and effective approach to chiral GC method development that speeds analysis while eliminating the need for slow and tedious manual interchange of columns. An example of developing a rapid isothermal GC method from the screening results obtained by the instrument is also shown.

A miniature mass spectrometer for liquid chromatography applications.

A miniature mass spectrometer capable of detecting analytes eluting from a high-performance liquid chromatography (HPLC) system is described and demonstrated for the first time. The entire instrument, including all pumps and the computer, is contained within a single enclosure that may be conveniently accommodated at the base of the HPLC stack. The microspray ion source, vacuum interface, ion guide, and quadrupole ion filter are all microengineered. These components are fabricated in batches using microelectromechanical systems (MEMS) techniques and considered to be consumables. When coupled to a standard HPLC system using an integrated passive split, the limit of detection for reserpine while scanning the full mass range is 5 ng on-column (1 pg of which is passed to the microspray). The mass range is m/z 100-800, and each spectrum is typically acquired at a rate of 1 scan per second.

Psychological treatments for irritable bowel syndrome: a systematic review and meta-analysis.

This study conducted a systematic review to assess the quality of existing literature on psychological treatments for irritable bowel syndrome and to quantify the evidence for their efficacy. Three independent reviewers (2 from England, 1 from the United States) coded the quality of 32 studies, 17 of which provided data suitable for meta-analysis. Meta-analysis of efficacy data (50% reduction of symptoms) gave an odds ratio of 12 (95% confidence interval = 5.56-25.96) and a number needed to treat of 2. Psychological treatments are, as a class of interventions, effective in reducing symptoms compared with a pooled group of control conditions. Questions regarding the relative superiority of specific psychological treatments and influence of active versus nonspecific treatment effects remain unanswered.